Second generation of non-coupled dinuclear copper sites modeled in Azurin.

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Second generation of non-coupled dinuclear copper sites modeled in Azurin.

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2011-05

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Protein design is a rapidly developing field of biological chemistry. Our approach to protein design involves layering structural components which are thought to be important for function onto a protein scaffold. In this way, we are uniquely able to individually test each layer for gain-of-function, as opposed to typical mutagenesis studies which look for loss-of-function. Elucidating structure and function relationships for copper proteins through protein design may ultimately lead to the design of industrial catalysts, inhibitors, or a further understanding of current biological pathways. Nitrite reductase (NiR) and peptidyl glycine -hydroxylating monooxygenase (PHM) both use non-coupled dinuclear copper sites to catalyze biologically relevant reactions; however mechanistic details of these enzymes are not fully understood. Azurin was chosen as a protein scaffold to model these non-coupled dinuclear copper sites due to its stability, high purification yields, and existing structural similarities to these enzymes. Azurin has an existing type one (T1) electron transfer copper site. A second copper binding site was incorporated to create the first generation models of these enzymes in azurin. These first generation models have shown catalytic activity but remain much less active than the native form. The catalyzed reactions require the transfer of an electron from a T1 copper site to the spectroscopically non-coupled type two (T2) copper catalytic site. Better facilitation of the electron transfer between these two sites is hypothesized to increase the activity in our models. Three second generation variants were created to facilitate electron transfer, as well as to provide insight into fundamental structure function relationships.

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University of Minnesota M.S. May 2011. Advisor: Steven M. Berry. Major: Chemistry. 1 computer file (PDF) vi, 84 pages.

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Bladholm, Erika Lee. (2011). Second generation of non-coupled dinuclear copper sites modeled in Azurin.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/131277.

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